This invention relates to methods for plant tissue culture and plant regeneration and in particular this invention relates to methods for transforming maize using Agrobacterium. 
Agrobacterium-mediated transformation methods have been used principally in dicotyledonous plants. Agrobacterium-mediated transformation in dicotyledons facilitates the delivery of larger pieces of heterologous nucleic acid as compared with other transformation methods such as particle bombardment, electroporation, polyethylene glycol-mediated transformation methods, and the like. In addition, Agrobacterium-mediated transformation appears to result in relatively few gene rearrangements and more typically results in the integration of low numbers of gene copies into the plant chromosome.
Monocotyledons are not a natural host of Agrobacterium. Although Agrobacterium-mediated transformation has been reported for asparagus (Bytebier B., et al. Proc. Natl. Acad Sci. USA 84:5354-5349, 1987) and for Dioscore bublifera (Schafer et al. Nature 327:529-532, 1987), it was generally believed that plants in the family Gramineae could not be transformed with Agrobacterium (Potrykus I. Biotechnology 8:535-543, 1990).
Grimsley et al. (Nature 325:177-179, 1987) reported that cDNA from maize streak virus could be delivered to maize plants by Agrobacterium tumefaciens and that the plants became infected with the virus. The research did not demonstrate that the cDNA reached the maize genome nor did it demonstrate stable integration of streak virus nucleic acid. Later studies demonstrated that Agrobacterium could be used to deliver a kanamycin-resistance gene and a GUS (xcex2-glucuronidase) gene to shoot apices of maize after shoot apex injury (Gould J. et al. Plant Physiol. 95:426-434, 1991 and U.S. Pat. No. 5,177,010 to Goldman et al.). In these studies plants generated from the tissue exposed to Agrobacterium contained both transformed cells and, non-transformed cells suggesting that the method did not uniformly deliver nucleic acid to the maize tissue.
European Patent Application Publication Number 604 662 A1 to Hiei et al. discloses a method for transforming monocotyledons using Agrobacterium. In this method, plant tissues were obtained from the monocotyledon maize and the tissues were exposed to Agrobacterium during the tissue dedifferentiation process. Hiei et al. disclose a maize transformation protocol using maize calli. Saito et al. disclose a method for transforming monocotyledons using the scutellum of immature embryos (European Application 672 752 A1). Ishida et al. also disclose a method specific for transforming maize by exposing immature embryos to A. tumefaciens (Nature Biotechnology, 1996, 14:745-750). The methods were optimized for inbred A188 maize lines. Transformation frequencies ranged from 12% to 30% at their highest for immature embryos from A188 lines that were 1.0-1.2 mm in length. Maize lines derived from crosses of A188 had significantly lower transformation frequencies ranging from 0.4% to about 5.3%. The transformation frequencies using A188 and A188 crosses are summarized in Table 1. A188 is not generally considered a commercially useful line and Ishida et al. failed to obtain recovery of stable transformants in lines other than those containing A188.
A need still exists for a method that will: (a) produce significantly higher transformation frequencies in lines other than those reported by Ishida et al. (supra); and, (b) produce transformed inbred lines other than line A188; including transformed inbreds representing a range of genetic diversities and having significant commercial utility.
This invention relates to methods for optimizing Agrobacterium-mediated transformation in maize. Significantly higher transformation frequencies for genotypes such as the product of A188 crossed to other inbreds would result in a higher throughput for production of transformed plants. This increased frequency would be useful, for example, to evaluate the efficacy of a larger number of genes in transgenic plants of corn or to generate a larger number of transgenic plants containing a particular foreign gene in a given period of time. Similarly, methods permitting the transformation of a variety of inbred lines would be commercially valuable.
In one aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating maize plants expressing the gene. In one embodiment the contacting step additionally comprises the step of contacting the immature embryos with Agrobacterium in a medium comprising N6 salts and in another embodiment the contacting step additionally comprises contacting the immature embryos with Agrobacterium in a medium comprising MS salts. Preferably the contacting step takes place in the absence of AgNO3. In one embodiment the embryos are cultured in a PHI basic media system and in another embodiment the embryos are cultured in a PHI combined media system. The immature embryos used in the method are preferably about 0.3 mm to about 4 mm in length and more preferably about 0.8 mm to about 2.0 mm in length. The Agrobacterium concentration used in the contacting step is preferably about 1xc3x97108 cfu/ml to about 1.5xc3x97109 cfu/ml and more preferably about 0.5xc3x97109 to about 1.0xc3x97109 cfu/ml. The contacting step preferably takes place in a liquid suspension and the co-cultivation step preferably takes place on a solid medium. Preferably, a medium containing MS salts is used in the regeneration step. In a preferred embodiment of this invention the method includes a resting step that comprises culturing the embryos in medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the embryos are cultured for about 1 to about 15 days. In one embodiment the antibiotic used is carbenicillin and a preferred concentration of carbenicillin is about 50 mg/l to about 250 mg/l. This method also relates to maize plants transformed by this method and to maize cells transformed by this method.
In another aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to said embryo in a medium comprising N6 salts; co-cultivating the embryo with Agrobacterium in a medium comprising N6 salts; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene in a medium comprising MS salts. Preferably, the medium of the contacting step lacks AgNO3 and the medium of the co-cultivating step includes AgNO3. Preferably the Agrobacterium concentration used in the contacting step is about 1xc3x97108 cfu/ml to about 1.5xc3x97109 cfu/ml. Preferably, the contacting step takes place in a liquid and the co-cultivating and culturing steps take place on a solid medium. In one embodiment of this method, the method additionally comprising the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the antibiotic is carbenicillin. This invention also relates to maize plants and to maize cells transformed by this method.
In yet another aspect of this invention, a method is disclosed for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to said embryo in a medium comprising N6 or MS salts; co-cultivating the embryo with Agrobacterium in a medium comprising MS salts; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene in a medium comprising MS salts. Preferably the medium of the contacting step lacks AgNO3 and the method of the co-cultivating step includes AgNO3. Also preferably, the contacting step takes place in a liquid and the co-cultivating and culturing steps take place on a solid medium. In one embodiment of this method, the method additionally comprising the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. 
This invention also relates to a method for optimizing the production of transgenic maize plants of a first genotype using Agrobacterium-mediated transformation comprising the steps of: isolating immature embryos from maize; separating the embryos into treatment groups; incubating each treatment group separately in a medium comprising N6 or MS salts and in a suspension of Agrobacterium at concentrations ranging from about 1xc3x97108 cfu/ml to about 1xc3x971010 cfu/ml; co-cultivating the embryos with Agrobacterium on a solid medium; culturing the embryos in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos transformed by Agrobacterium; identifying the treatment group with the highest transformation frequency ; and using the concentration of Agrobacterium generating the highest transformation frequency to transform other embryos from the first genotype. In one embodiment of this method, the medium of the incubating step and the co-cultivating step is a medium comprising N6 salts and in another embodiment of this method, the medium of the incubating step is a medium comprising MS salts and the medium of the co-cultivating step is a medium comprising N6 salts. In yet another embodiment, medium of the incubating step is a medium comprising N6 salts and the medium of the co-cultivating step is a medium comprising MS salts. The method also preferably includes the step of resting the embryo by culturing the embryo in a medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. Preferably the antibiotic is carbenicillin and preferably, the combined length of the co-cultivating step and the resting step is at least three days. Where a resting step is used, the length of the resting step is from more than 0 to about 10 days. In a preferred embodiment, the length of the resting step is about 3 to about 5 days.
In another aspect of this invention, the invention relates to transformed maize plants produced by a method comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene.
In yet another aspect of this invention, the invention relates to transformed maize cells produced by a method comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; and culturing the embryo in a medium comprising N6 salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene: and identifying embryos expressing the gene.
In a preferred aspect of this invention, the invention relates to a method for transforming maize using Agrobacterium comprising the steps of: contacting at least one immature embryo from a maize plant with Agrobacterium capable of transferring at least one gene to the embryo; co-cultivating the embryo with Agrobacterium; culturing the embryo in a medium containing salts other than MS salts, an antibiotic capable of inhibiting the growth of Agrobacterium, and a selective agent to select for embryos expressing the gene; and regenerating plants expressing the gene.